This invention relates to a method for the culture of cells and, more particularly, to the submerged culture of animal cells using microcarriers for the attachment of cells in suspension.
In recent years there has been rapid growth in the development of various methods for the culturing of cells in suspension. The attainment of high cell densities is a primary objective of many of these approaches. The use of a cell culture vessel with controlled agitation by means of a magnetic stirrer bar or a mechanically driven impeller on a shaft is a typical feature of these methods. Examples of such apparatus are disclosed in U.S. Pat. Nos. 2,958,517; 3,639,932; 3,572,651, 3,622,122; and 3,649,465. These are essentially batch type spin culture devices or spinner flasks in which the cells are incubated in a fixed amount of nutrient under appropriate culture conditions until cell growth has ceased.
Continous cell culture systems and apparatus also have been described heretofore in which fresh culture medium can be added and spent medium can be separated from the growing cells by filtration and withdrawn from the flask on a continuous or semi-continuous basis as seen from U.S. Pat. Nos. 4,166,768 and 4,178,209.
While ordinary suspension cultures are suitable for growth of certain mammalian cell lines, other cells, and particularly human diploid cells, are anchorage-dependent in that they require support surface means for cell attachment. Examples of such cell culture systems are the monolayer growth systems in T-flasks, roller bottles, artificial capillary propagators and multi-plate propagators.
In order to provide the advantages of large scale suspension culture with provision for cell attachment, microcarrier systems have been developed. The successful use of microcarriers for cell culture was first reported by van Wezel, Nature 216, 64-65 (1967). The method of van Wezel consisted of growing cells as monolayers on the surface of positively charged DEAE-Sephadex.RTM. beads (grade A-50, about 100.mu. diameter) suspended in culture media in a stirred vessel. The stirred vessel used by van Wezel was the Bilthoven microbial culture unit described by van Hemert, Biotechnol. Bioeng. VI, 381-401 (1964). In this method, different cell lines, human diploid cells, and primary rabbit kidney cells were successfully cultivated. The production of polio virus in the microcarrier culture was examined by van Wezel and the virus multiplication was found to be essentially similar to that in monolayer culture. Further description of that microcarrier system is disclosed by van Wezel et al., Process Biochem., March 1978, pp. 6-8 and 28, wherein it is stated that as far as cultivation of very sensitive cell types, such as human diploid cell strains is concerned, the system is still not completely satisfactory.
A modification of the van Wezel method is described by Levine et al, Somatic Cell Genetics 3, 149-155 (1977) and U.S. Pat. Nos. 4,036,693 and 4,189,534. This system uses essentially a spinner flask with a magnetically driven stirrer bar. Some success with this system at a small scale of 100 ml working volume has been reported by Levine et al., Biotechnol. Bioeng. 21, 821-45 (1979). However, other investigators have cast doubt upon the applicability of this system to larger scale use with human diploid cells; Lewis and Volkers, Develop. Biol. Standard. 42, 147-151 (1979).
In copending application Ser. No. 161,614, filed June 20, 1980, and now U.S. Pat. No. 428,854 and assigned to a common assignee, an improved cell culture system and apparatus is disclosed which is particularly useful for adaptation to cell culture on microcarriers in agitated suspension media. The agitator has flexible sheets which provide a gentle agitation for sensitive cells and fragile microcarriers.